RESEARCH ARTICLE Open Access

The life of the freshwater bryozoanStephanella hina (Bryozoa,Phylactolaemata)—a crucial key toelucidating bryozoan evolutionThomas Schwaha1* , Masato Hirose2 and Andreas Wanninger1

Abstract

Background: Phylactolaemata is the earliest branch and the sister group to all extant bryozoans. It is considered asmall relict group that, perhaps due to the invasion of freshwater, has retained ancestral features. Reconstruction ofthe ground pattern of Phylactolaemata is thus essential for reconstructing the ground pattern of all Bryozoa, andfor inferring phylogenetic relationships to possible sister taxa. It is well known that Stephanella hina, the solemember of the family Stephanelllidae, is probably one of the earliest offshoots among the Phylactolaemata andshows some morphological peculiarities. However, key aspects of its biology are largely unknown. The aim of thepresent study was to analyze live specimens of this species, in order to both document its behavior and describe itscolony morphology.

Results: The colony morphology of Stephanella hina consists of zooidal arrangements with lateral budding sitesreminiscent of other bryozoan taxa, i.e., Steno- and Gymnolaemata. Zooids protrude vertically from the substrateand are covered in a non-rigid jelly-like ectocyst. The latter is a transparent, sticky hull that for the most part showsno distinct connection to the endocyst. Interestingly, individual zooids can be readily separated from the rest of thecolony. The loose tube-like ectocyst can be removed from the animals that produces individuals that are unable toretract their lophophore, but merely shorten their trunk by contraction of the retractor muscles.

Conclusions: These observations indicate that S. hina is unique among Phylactolaemata and support the notionthat bryozoans evolved from worm-like ancestors. In addition, we raise several arguments for its placement into aseparate family, Stephanellidae, rather than among the Plumatellidae, as previously suggested.

Keywords: Ectoprocta, Stephanellidae, Phylactolaemata, Ectocyst evolution, Solitary zooids

BackgroundBryozoa or Ectoprocta is a group of sessile, colonialfilter-feeders of approximately 6000 extant species. Itsphylogenetic relationship to other lophotrochozoan taxaremains controversial (e.g. [1–4]). Due to shared mor-phological features in their general organization, includ-ing their feeding apparatus, the lophophore, this groupwas traditionally united with the Brachiopoda and Phor-onida as ‘Lophophorata’ or ‘Tentaculata’ [5]. However,

many molecular phylogenies have failed to support the‘Lophophorata’ concept. Most have supported instead aclose relationship between phoronids and brachiopods(e.g. [3, 6]), whereas only few recent studies have lentsupport to the ‘lophophorate’ grouping [7–9].Bryozoans typically consist of several individuals,

called zooids, which constitute the colony. Each zooid istypically divided into the cystid, which constitutes thebody-wall, which is fortified with different extracellularsecretions, and the polypide, which consists of the soft-body parts. Bryozoa can be divided into three class-leveltaxa: Phylactolaemata, Stenolaemata and Gymnolaemata.Morphological and molecular analyses agree that

* Correspondence: thomas.schwaha@univie.ac.at1Department of Integrative Zoology, University of Vienna, Althanstraße 14,Vienna 1090, AustriaFull list of author information is available at the end of the article

© The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Schwaha et al. Zoological Letters (2016) 2:25 DOI 10.1186/s40851-016-0060-5

Phylactolaemata is the basal-most branch and sister-group to the remaining taxa [10–12]. Phylactolaemata isa small group of approximately 80 species worldwidewithin six families that only occur in freshwater habitats[13]. Their members show different variations of theectocyst, which may be either chitinous or gelatinous.Various analyses agree that Stephanellidae with the solecurrently recognized species Stephanella hina Oka, 1908is one of the earliest branches among the Phylactolae-mata [14]. This species is almost exclusively reportedfrom Japan and Southeast Asia [15, 16] and the eastcoast of North America [17], and it shows a unique col-ony morphology with stolon-like connections and lateralbudding loci [18]. In addition, its statoblasts and in par-ticular the formation of the sessoblasts seems to differfrom all other phylactolaemate bryozoans [19]. This spe-cies is also unique in that it starts to grow in Decemberand starts to decay in mid-April when other phylactolae-mates start to thrive after overwintering [20]. Only fewother species are known to survive cold winter tempera-tures as adults [13].Surprisingly, almost no data are currently available

for this species; even photographic documentation isalmost entirely lacking [16, 18, 21]. In addition,whether Stephanella constitutes a separate family-level taxon or is part of the Plumatellidae, the largestof all phylactolaemate families, remains unclear. Ouraim in the present study was to identify and documentthe biology of this neglected species. As the earliestbranch, the reconstruction of the phylactolaematebody plan may yield essential information into theevolution of the whole phylum, as well as the evolu-tion of coloniality among metazoans. Bryozoa is theonly animal phylum in which the members are allcolonial. However, since all other possible lophotro-chozoan outgroups have a solitary ground pattern, it isclear that the pre-bryozoan ancestor was also asolitary, probably worm-shaped, organism. Hence theelucidation of the phylactolaemate morphologicalground pattern could yield insight into the evolutionof solitary into colonial forms.

MethodsSamples were collected in April 2014 and February 2015and 2016 in Japan, either in the pond of the campus ofthe University of Tsukuba or in a freshwater pond inTsuchiura-shi in close vicinity to the campus. Sampleswere collected from mainly wooden substrates andtransferred into the laboratory for further inspection andtreatment. Plants and artificial substrates (mainly gar-bage, shoes, etc.) were not colonized by any specimens.Living specimens were documented and filmed with aNikon J1 camera (Nikon, Tokyo, Japan) mounted on ac-mount on a Nikon SMZ 1500.

Results & discussionGeneral structure of the colony and zooidsStephanella hina colonies form an array of intercon-nected zooids, with each zooid generally having up tofour connections to other zooids within the colony(Fig. 1a and b). These interconnections are restricted tothe substrate and are, particularly in older colonies,stolon-like. Younger colonies commonly show a widerinterzooidal connection. This corresponds to previousdescriptions [18, 21] and is unique for a phylactolaematebryozoan where zooids are normally interconnected bymuch wider spaces than in Stephanella. In this respect asimilar tube-like connection is only found in the pluma-tellid genus Stolella [22, 23]. However, in the latter theseare not as pronounced as in S. hina. Since there arethree budding loci—one distal and two lateral—a total offour stolon-like connections are present in S. hina(Fig. 1a, b). Lateral buds are not present in any otherphylactolaemate. This simple runner-like colony shape isalso characteristic of early fossil representatives as well

Fig. 1 Budding sites of Stephanella hina. a View of an elongated distalbud, with two lateral buds and developing statoblasts. b Parts of aretracted colony, showing the cruciform growth of the colonies due tolateral budding. c Close-up of the basal part of a detached, single zooid,showing an early two-layered bud at its base close to the retractor muscle.Abbreviations: ab – advanced bud, b – bud, bs – basal attachment side,cae – caecum, ds – developing statoblasts, en – endocyst, lb – lateralbuds, rm – retractor muscle, s – stolon, z – zooid

Schwaha et al. Zoological Letters (2016) 2:25 Page 2 of 10

as recent, probably early branching, taxa of the othertwo bryozoan taxa Stenolaemata (e.g. Corynotrypidae)[24–26] and Gymnolaemata (e.g. Arachnidium and Car-doarachnidium in Ctenostomata [27]; e.g. Pyripora andPyriporopsis in Cheilostomata [28, 29]). Considering theprimitive pattern of budding in Stephanella as an earlybranch of Phylactolaemata [14], it is possible that it hasretained the early or the original budding mode, and thatother Phylactolaemata have reduced it.The main part of each zooid extends vertically from

the substrate as a long slender tube that carries all themajor components of the polypide, i.e., the digestivetract and the tentacle crown, the lophophore, which isused for filter-feeding (Figs. 2 and 3, see also Additionalfile 1: Video S1). From the lophophoral base, the

intertentacular membrane extends towards the abfrontalside of the tentacles. It is rather short and surrounds thewhole lophophore equally (Fig. 4b and d, see Additionalfile 2: Video S2). The tentacles, the epistome and thedistal part of the pharynx show a reddish-brownish hue,which is more pronounced when animals are retractedand tissues are much more compact (Fig. 4a, b and d,see Additional file 2: Video S2, Additional file 3: VideoS3, Additional file 4: Video S4). This coloration has beenpreviously described by several authors [16, 18, 21]. Thetentacle tinge is reportedly lost after two days of culturein pure tap water [16]. The digestive tract consists of thecommon parts found in other bryozoans, i.e. pharynx,oesophagus, cardia, caecum, intestine and anus. In oursamples the digestive tract showed a blue-greencoloration in most cases (Additional file 3: Video S3,Additional file 4: Video S4, Additional file 5: Video S5).The color of the digestive tract is commonly influencedby the food source, but the exact food contents have notbeen analyzed so far in S. hina. Previously, the color-ation of the stomach was described as yellowish-green

Fig. 2 Comparison of the colony of Stephanella hina in and out ofthe water, showing the non-supportive and flexible ectocyst. aStephanella hina colony on a piece of wood taken out of the water.Note the collapsed appearance of the colony with the zooids lyingflat on the substrate. b Piece of a colony submerged into water.Note the upright position of the zooids extending vertically fromthe substrate. Abbreviations: ec – ectocyst, z – zooid

Fig. 3 General morphology of colonies of Stephanella hina. a Lateralview of a colony, showing several individual zooids extending theirlophophores into the water column. Note that the polypides extendvertically from the substrate. The transparent ectocyst can only bediscerned by particles adhering to it. b Top view of a colony showingthe arrangement of the individual zooids within the colony and thetypical horseshoe-shaped lophophore. Abbreviations: ec- ectocyst,l – lophophore, rz – retracted zooid

Schwaha et al. Zoological Letters (2016) 2:25 Page 3 of 10

[21] to pale brown [18]. Retraction of the polypide iseffected by a paired retractor muscle that emanates fromthe basal side and inserts at the lophophoral base andparts of the digestive tract (Figs. 1c and 4c, seeAdditional file 3: Video S3, Additional file 4: Video S4,Additional file 5: Video S5, Additional file 6: Video S6).The general transparency of the animals allows readyobservation of the contraction and relaxation of thesemuscles. Contraction of the regular body wall muscula-ture increases pressure within the coelomic cavity andalong with relaxation of retractor and apertural musclespushes the polypide out of the cystid. The body wallmusculature can be observed in live specimens (Fig. 5a).The funicular cord runs from the proximal pole of thecaecum to the body and carries the anlagen or fully dif-ferentiated free-floating statoblasts, the floatoblasts(Fig. 6). Floatoblasts in S. hina are circular in shape(Fig. 6a–c). At the basal side, sessoblasts are commonlyattached to the substrate (Fig. 6d and e). These oftenoccur in certain interzooidal widenings of the stolonal

network, and rarely exceed 2–3 in number. The situ-ation thus resembles that of other phylactolaemate bryo-zoans [13, 30]. So far, no gonads of either sex have beenencountered. A single previous description found well-developed testes and some growing ovaries in veryyoung colonies in December at 5 °C water temperature,but could not follow its subsequent development due tofrozen ponds and lakes [20].

The ‘cystid’ of Stephanella hinaThe vertically extending tubes containing the polypidesare embedded into a copious amount of the jelly-like,transparent ectocyst. Two different variations of thisectocyst were encountered: either it forms a connectingmass between the extended polypides, or each polypideis surrounded by its own tube, which is not connectedto neighbouring ectocysts. Particularly in the latter condi-tion, the cystid is not a firm structure that maintains thegeneral structure of the colony, but appears to be morebuoyant (Figs. 2b and 3, Additional file 1: Video S1). This

Fig. 4 Details of a single zooid of Stephanella hina. a View of the lophophore with the horseshoe-shaped tentacle arrangement and the epistomeprotruding over the mouth opening. Note the orange tinge of the epistome. b Lateral view of the lophophoral base, showing the evident coloration ofthe epistome as well as the pharynx. Note the intertentacular membrane on the outer side of the lophophore. c Lateral view through the transparentectocyst showing parts of the digestive tract as well as retractor muscle strands traversing the visceral coelom. d View from the basal side of the lophophoreand the intertentacular membrane extending from the lophophoral base towards the outer area of the tentacles. Abbreviations: cae – caecum, en . endocyst,ep – epistome, int – intestine, itm – intertentacular membrane, l – lophophore, ph – pharynx, rm – retractor muscle, t – tentacle, ts – tentacle sheath

Schwaha et al. Zoological Letters (2016) 2:25 Page 4 of 10

is most evident when colonies and their substrate are re-moved from the water, which results in a collapse of thecolony, resulting in a flattened appearance (Fig. 2a). Thesoft jelly-like and barely self-supportive form of this ecto-cyst was previously recognized [17, 18], whereas the firstmore dense form has not been reported. No distinct envir-onmental cues are known for different cystid structureamong colonies, especially since colonies were collectedfrom the same localities and sometimes also at the sametime of the year.As described above, the ectocyst is for the most part

transparent – in general, however, this ‘cystid’ has stickyproperties and particles floating in the water columneasily adhere to it as previously described by Oka [21](Figs. 2b and 3a; Additional file 3: Video S3). Cystidstructures in the non-calcified Phylactolaemata are eithergelatinous as in most of the larger forms (Pectinatellidae,Cristatelllidae, Lophopodidae) or sand-encrusted to

chitinous in the smaller, rather branching representatives(Plumatellidae, Fredericellidae) [13, 31]. The ectocyst inS. hina is different from that of the above-mentionedtaxa, and is probably best characterized as jelly-like. It ismost comparable in appearance and mechanical pro-perties to the protein/glycoprotein jelly-coats of frogembryos [32].

Fig. 5 Coelomic structures of Stephanella hina. a Lateral view of asolitary zooid of Stephanella hina, showing body-wall musculature aswell as coelomocytes within the coelomic cavity. b Lateral view ofthe proximal part of a zooid, showing the regular arrangement ofciliary fields of the peritoneal lining of the coelomic cavity as well asstrands of retractor muscle fibres traversing the same. Abbreviations:bm – body-wall musculature, cae – caecum, cc – coelomocyte,cf – ciliary fields of ther peritoneal epithelium, rm – retractor musclefibres, s - stolon

Fig. 6 Statoblasts of Stephanella hina. a Statoblasts and developingstatoblasts in a retracted zooid. Note the distance between the jelly-likecystid and the zooid (double arrow). b Single zooid with 14+ floatoblastsin the proximal cavity behind the zooid. c Detail of expulsed floatoblastson the water surface. The floatoblasts are circular and show a darkerannulus for floating and an inner orange fenestra, which contains thegermination mass. Note a darker spot/central protuberance in the middlefloatoblast indicating the deutoplasmatic side (ventral valve) of thestatoblast. d Sessoblasts located in a distinct part of the colony notdirectly associated with a zooid but merely with stolons. The stolon-likeprocesses interconnect individual zooids of the colony. e Detail of asessoblast. The sessoblast is firmly attached to the substrate andlacks a floating annulus. Note on the left side sessoblast-remains. Inthe latter only the attaching cementus is present, indicating theprobability that the frontal valve of the sessoblast was mechanicallyremoved. Abbreviations: cp – central protuberance, ds – developingstatoblast, ec – ectocyst, fb – floatoblast, l – lophophore, oc – openingof the ectocyst tube, s – stolon, sb – sessoblast, z –zooid

Schwaha et al. Zoological Letters (2016) 2:25 Page 5 of 10

In all bryozoan zooids the cystid consists of an inner,living endocyst (the body-wall with the outer epidermisand inner peritoneum) and an extracellularly secretedcuticle, the ectocyst or sometimes called periostracum(see eg. [33] for phylactolaemates and [34] for calcareoustaxa). These two components are commonly attachedfirmly to each other, especially in the calcified Stenolae-mata and Cheilostomata. Analysis of the retractionprocess in Stephanella hina shows that the endocyst isfor the most part not attached to the ectocyst and thusactually not a cuticle of the endocyst but a tube sur-rounding the zooid (Fig. 7). During the retractionprocess, the endocyst is quite distant from the ectocyst,leaving a considerable gap (Fig. 7, Additional file 7:Video S7). This kind of ectocyst is thus unique amongPhylactolaemata and Bryozoa in general and superficiallyresembles tubes of other filter-feeding lophotrochozoans(e.g., tube-forming polychaete annelids and phoronids).

CoelomocytesDue to its transparency, internal structures can readilybe observed. One very obvious feature in Stephanellahina is commonly elongated to roundish coelomocytesin the body cavity of the animals (Fig. 5a, see Additionalfile 6: Video S6, Additional file 8: Video S8 andAdditional file 9: Video S9). Their amount and densityseem to be highly variable and range from very few toseveral dozens. Cues affecting their abundance are un-known. Ciliary tufts regularly situated at the inner peri-toneal layer (cf. [30, 33]) create a current in the

coelomic fluid that circulates the coelomocytes withinthe coelomic cavity (Fig. 5b). Previously, up to nine dif-ferent coelomocytes were categorized in the Phylactolae-mata [35]. None have been described as elongated/rod-like cells as found in S. hina (see Additional file 6: VideoS6, Additional file 8: Video S8 and Additional file 9:Video S9). Coelomocytes in bryozoans appear to play dif-ferent important roles that remain poorly understood.They appear to originate as detaching cells from the peri-toneal layer of the body-wall. Especially vital dye experi-ments indicate that some of these cells have excretoryfunctions [35–37], whereas some were commonly termed‘leucocytes’ or ‘lymphocytes’. An immunological function,however, could not be proven so far. Particular excretoryorgans/osmoregulatory organs such as proto- or metane-phridial systems are absent among bryozoans (cf. [30]);their function seems to be taken over by coelomocytes, ashas been described for marine bryozoans [38, 39].In the current investigation, a few cases of the expulsion

of coelomocytes were observed from specimens contain-ing numerous coelomocytes (Additional file 10: VideoS10). This phenomenon was previously documented in afew other phylactolaemates [40–42] and occurs via thevestibular pore at the anal side of the vestibular wall at theorifice. The vestibular pore is a not well-recognized struc-ture which in addition to the expulsion of coelomocytes isalso the point where statoblasts as well as sperm are com-monly liberated [13, 23, 40, 42–44]. Only recently the porewas morphologically confirmed in the phylactolaemateLophopus crystallinus. In the latter, muscles are present at

Fig. 7 The retraction process of an individual zooid of Stephanella hina in regard to the ectocyst. a Completely retracted zooid. b Mid-retracted/extended zooid and c protruded zooid. Note that in all images there is a distinct gap between the endocyst and the ectocyst, and that theendocyst slides past the ectocyst during the retraction process. Abbreviations: as – cup-shaped attachment site, ca – caecum, ec – ectocyst,en – endocyst, fb – floatoblast, int – intestine, l – lophophore, o – orifice, oc – opening of the ectocyst tube

Schwaha et al. Zoological Letters (2016) 2:25 Page 6 of 10

the pore that enable active closure after the release of sub-stances [42]. It is highly likely that S. hina and other phy-lactolaemate species possess such a pore, but this needs tobe confirmed morphologically on a broader scale. Gymno-laemate bryozoans possess a comparable pore, the supra-neural pore, which seems, at least partially, to fulfillsimilar functions. However, it is located at the lophophoralbase rather than in the vestibular wall [45].

Experimental observationsDe-colonisation of coloniesSome of the sample material contained single zooids notconnected with others; these were thus clearly solitary.Later observations showed that this resulted mostly fromthe disruption of the stolon during removal of the zooidsfrom the substrate. Consequently, individual zooids wereexcised from the colony to observe whether this wouldaffect the life of the individuals. We found that de-colonisation does not seem to have any negative effect.Separated zooids that contain fully differentiated floato-blasts generally float on the top of the water column.Considering that separation of the individuals or a col-

ony did not seem to affect the future life of the coloniesraises questions about how well zooids are integrated intocolonial communication. For most bryozoans, colonial in-tegration of the individuals is assumed (i.e. the assumptionis that individuals communicate in some way with otherzooids of the colony, particularly in response to threatssuch as predators). For this purpose a colonial nervoussystem is present within the body-wall (see [46]). In themore advanced and integrated taxa Stenolaemata andGymnolaemata, the colonial plexus is localized in moredirect patterns than in the Phylactolaemata, in which thenervous system in the body-wall consists merely of a dif-fuse plexus [30, 46–49]. Since information on the nervoussystem in Stephanella hina is lacking, the extent of neur-onal integration of zooids is not known, but it is clear thatzooids communicate by stolon-like connections acrosslonger distances than in other freshwater bryozoans. Nu-merous observations in which individual zooids weretapped to provoke retraction found no effect on neighbor-ing zooids (see Additional file 11: Video S11). This is notuncommon, especially for freshwater bryozoans, wherestimulation of individual zooids does not necessarily leadto the retraction of more than just the affected zooid. In-stead, (mechanical) stimulation of the cystid wall resultsin simultaneous retraction [50]. Due to the lack of a firmconnection of the soft ectocyst to the endocyst, cystid vi-brations often do not result in a colonial retractionprocess. Also, in some cases removing individual zooidsfrom the colony did not result in any defensive responsein other zooids of the colony, whereas wounding has beenfound to be a common stimulus for retraction in otherphylactolaemates [50]. Suffice to say, this appears to be a

rather ineffective protective mechanism. Moreover, S. hinacolonies commonly do not react to regular shifts in thewater column; regular movement of the dishes containingthe colonies did not cause the zooids to retract, but rathercaused the vertically standing tube-like extensions of thezooids, including the jelly-like ectocyst, to follow the mo-tion of the water. This gave the colony the appearance of‘headbanging’ tentacle crowns (see Additional file 12: VideoS12). Currently, reduced predation during the cold monthsappears to be the only explanation for why zooids appearrather unresponsive to such stimuli. As aforementioned, S.hina is one of the few species that occurs in winter. Perhapslower temperatures also lead to a lower metabolism inthese animals, and thus slower response times. Alterna-tively, or additionally, predation risk may be significantlydecreased during colder times in freshwater.

Ectocyst removal and cystid evolutionAs described above, the ectocyst is for the most part notfirmly attached to the body-wall; nonetheless, the retrac-tion process functions properly. Investigation of de-colonized, solitary forms shows that parts of the basalwall form a cup-like structure that connects with theectocyst. This is the area at which the retractor muscle fi-bres attach, and thus represents the support for the retrac-tion process. This limited attachment of the body-wall tothe ectocyst gave the opportunity to release zooids fromtheir ectocyst hull (Additional file 13: Video S13). Severingthe attachment site, in most cases with a pair of forceps,gave rise to zooids devoid of an ectocyst. The animalsreadily survived this treatment and continued feeding.Tapping animals that lack an ectocyst does not cause re-traction of the tentacle crown, only a longitudinal contrac-tion of the trunk as, in such individuals, the retractormuscle has no support from the area normally attached tothe substrate (Additional file 14: Video S14). However,such animals are capable of restoring/regenerating theirectocyst coating. At first, the new ectocyst is produced onthe basal site at the attachment of the retractor fibres inorder to enable the typical retraction process. The ectocystrenewal proceeds distally towards the tentacle crown thatprotrudes into the water column. Mechanical stimulationof such specimens leads to partial contraction of the trunk(Additional file 15: Video S15).Experimental ectocyst removal was previously con-

ducted only in the lophopodid L. crystallinus [51], inwhich colonies also survived the procedure without greatharm. The ectocyst in L. crystallinus in general is just athin gelatinous layer that is in rather loose connectionwith the endocyst [40]. With the current position of ste-phanellids and lophopodids, this may imply that the ori-ginal ectocyst was not a proper cuticle attached to thebody-wall, but merely a tube-like, semi-adhering protect-ive secretion. The lophopodid genus Lophopodella

Schwaha et al. Zoological Letters (2016) 2:25 Page 7 of 10

commonly has a thin layer of ectocyst (Hirose pers. obs.),whereas the condition among the other lophopodid genusAsajirella hast not been analyzed recently. In the latter itappears to be more massive and gelatinous [52, 53]. It isdefinitely present in copious amounts on the basal side(the side facing the substrate), but it is not clear to whatextent it is present on the frontal side facing the water.Pectinatella magnifica (sole species of Pectinatellidae) alsoproduces copious amounts of gelatinous ectocyst, but onlythe basal side is covered by ectocyst, whereas the frontalone is naked [52]. Likewise, Cristatella mucedo (sole rep-resentative of the Cristatellidae) also produces only a thinectocyst at its creeping sole towards the basal side [51,52]. Pectinatellidae and Cristatellidae are generallyregarded as closely related and probably constitute sister-groups [14]. Cristatella has motile colonies through thewhole life-stages, whereas Pectinatella colonies are sessilegelatinous masses and only young colonies are motile[54–56]. Consequently, the three phylactolaemate familiesLophopodidae, Cristatellidae and Pectinatellidae are mo-tile, at least at one point during their ontogeny [56]. Arigid and firm ectocyst is thus obstructive for any move-ment. It appears that only in the most speciose family, thePlumatellidae, the separation of the ectocyst is not pos-sible; the same applies to the Fredericellidae. However, inthe latter it should be noted that zooids are capable ofdetaching from the cystid and leave the colony in form ofswimming zooids under unfavorable conditions [57].Given that the latter two families are currently consideredas the derived form [14], it is likely that the phylactolae-mate ancestor was a semi-motile, gelatinous animal with arather loose ectocyst. Stephanella hina appears to besomewhat intermediate between a motile and strictly ses-sile form, since it is definitely sessile, but almost shows noconnection to the loose ectocyst.

The StephanellidaeThe present study confirms that Stephanella hina is indeeda unique species among the Phylactolaemata. With its typ-ical features, such as the colony structure, the jelly-likeectocyst and the mostly missing connection between endo-and ectocyst, it is clear that this species cannot be assignedto the Plumatellidae as it has been in previous studies (e.g.[17, 58]), but merits a separate family Stephanellidae as ori-ginally proposed by Lacourt [15]. Besides the clear distin-guishing features presented above, Stephanellidae is,together with the Plumatellidae, the only phylactolaematefamily to produce sessoblasts (sessile dormant stages/stato-blasts). However, the formation of the sessoblast is essen-tially different in S. hina than in all plumatellids studied[18, 19]. In plumatellids the basal side of the sessoblast, at-tached to the substrate, derives from the so-called cysti-genic side, whereas the opposite condition with thedeutoplasmatic side forming the basal side is found in S.

hina [18]. This substantiates separate placement into theStephanellidae and indicates that sessoblasts probablyevolved twice within the phylactolaemates. Finally, Frederi-cellidae and Plumatellidae show an oro-median gap withinthe intertentacular membrane [40], which all other families,including Stephanella, lack. The functional significance ofthis gap is not clearly understood. The lack of this particu-larly neglected feature again supports the placement ofStephanella into a separate family-level taxon. Accordingly,the features defining Stephanellidae are (i) stolon-likegrowth pattern with polypides mostly extending verticallyfrom the substrate, (ii) lateral budding loci in addition tothe normal buds produced on the oral side as in other Phy-lactolaemata, (iii) sessoblasts attaching to the substrate withthe deutoplasmatic side, (iv) floatoblast circular and notoval as in Plumatellidae, (v) jelly-like ectocyst for the mostpart not in direct connection to the endocyst, (vi) lack oforo-median gap in the intertentacular membrane.

Conclusions and outlookStephanella hina is an ideal candidate for experimentalapproaches, as the transparency of the ectocyst as wellas the easy detachment of single individuals from thecolony allows insight into internal processes, such asdigestion and the enigmatic coelomocytes. Other phylac-tolaemate families, such as the Lophopodidae, Cristatel-lidae or Pectinatellidae, are also transparent, but theirlarge size and compact colony shape impede propermicroscopic investigation. Moreover, they are difficult tokeep in the laboratory. We kept large Stephanella col-onies in a simple, aerated bucket at room temperature inthe lab without any special care for over a week. Thecolonies were still in very good condition, which indi-cates that laboratory culture should be possible withouttoo much effort. Some of the smaller branching forms,such as plumatellids or fredericellids, are opaque in theirnative habitat, but in case of Fredericella can be ren-dered transparent under proper laboratory culture [59].The latter is particularly interesting for use in parasito-logical investigations, since phylactolaemate bryozoansare definitive hosts of myxozoan parasites, which can beobserved within the body cavity [60]. So far no parasiticstages are known for Stephanella, a fact that perhapscould be correlated to their life in the cold times of theyear, but it might be an interesting aspect to study in thefuture. Some undulating worm-like objects were encoun-tered in some zooids. Some of these appear to be slowlycirculating elongated coelomocytes, whereas others mightrepresent ingested particles that were not properlydigested and traverse the digestive tract unharmed.What potential does this species have for phylogenetic

inferences of the Bryozoa? Taking this species as one ofthe earliest branches, if not the earliest branch among

Schwaha et al. Zoological Letters (2016) 2:25 Page 8 of 10

Phylactolaemata, the conditions found in S. hina are per-haps plesiomorphic. Bryozoa were often regarded beingderived from worm-shaped organisms, perhaps similarto the Phoronida (e.g. [61]). Many filter-feeding annelidsand also phoronids are capable of secreting protectivetubes from the body-wall which the animals can retractinto ([62–64] for Annelida) ([65] for Phoronida). Theectocyst of S. hina is more similar to these tubes ratherthan the firmly attached ectocyst (thus a proper cuticle)of other bryozoans. Filter-feeding worms that live intubes commonly use the longitudinal musculature to re-tract into the tubes. Accordingly, where has the retractormuscle of bryozoans evolved from? The last attempt ofan evolutionary interpretation regarded it as conden-sation and separation of longitudinal body-wall mus-cles (cf. [66]). This view was, however, biased by the‘lophophorate’ concept, and took the phoronids as closestto the Bryozoa for granted. In this line of thought, the der-ivation of the retractor muscle appears likely from theprominent feather-like longitudinal muscle bundles foundin phoronids. However, even without this bias, the deriv-ation of the retractor muscle from a longitudinal body-wallmusculature appears likely when a worm-shaped ancestoris assumed. The condition of zooids without an ectocyst inStephanella hina also showed trunk contractions, similarto the shortening of a worm-shaped body due to contrac-tion of a longitudinal body-wall musculature.In the early branching lophopodids an epistome is

lacking [33], which is traditionally considered to be onethe phylactolaemate-specific characters (e.g. [10, 30]).Such a small flap-like protuberance over the mouthopening is also present in other ‘lophophorate’ phylaand was considered homologous among the three‘lophophorates’. Future studies should deal with thestructure of different organ systems such as the epistomein order to assess whether this character has indeedbeen present at the base of all Phylactolaemata or hasevolved within this group. In addition, future field workshould aim at finding sexually mature colonies as well aslarvae, since any kind of sexual reproduction is currentlyunknown in Stephanella.

AcknowledgementsSpecial thanks to all the departmental members of the AORI, KashiwaCampus of the University of Tokyo for their kindness and hospitality.

FundingSpecial thanks to the Faculty of Life Sciences and the Dean’s office forfinancial support to TS for travelling to Japan.

Availability of data and materialsThe datasets during and/or analysed during the current study available fromthe corresponding author on reasonable request.

Authors’ contributionsTS carried out the experiments and wrote the draft. All authors contributedto the writing of the manuscript. MH coordinated necessary field work and

lab work in Japan as well as contributed to data interpretation. AWsupervised the study and contributed to data interpretation. All authors readand approved the final version of the manuscript.

Competing interestsThe authors declare that they have no competing interests.

Consent for publicationNot applicable.

Ethics approval and consent to participateEthical approval and consent to participate were not required for this work.

Author details1Department of Integrative Zoology, University of Vienna, Althanstraße 14,Vienna 1090, Austria. 2Atmosphere and Ocean Research Institute, Universityof Tokyo, 5-1-5 Kashiwanoha, Kashiwa-shi, Chiba 277-8564, Japan.

Received: 7 September 2016 Accepted: 16 November 2016

References1. Halanych KM. The new view of animal phylogeny. Annu Rev Ecol Evol Syst.

2004;35:229–56.2. Dunn CW, Hejnol A, Matus DQ, Pang K, Browne WE, Smith SA, Seaver E,

Rouse GW, Obst M, Edgecombe GD, et al. Broad phylogenomic samplingimproves resolution of the animal tree of life. Nature. 2008;452:745–U745.

3. Hejnol A, Obst M, Stamatakis A, Ott M, Rouse GW, Edgecombe GD, Martinez P,Baguna J, Bailly X, Jondelius U, et al. Assessing the root of bilaterian animalswith scalable phylogenomic methods. Proc R Soc B Biol Sci. 2009;276:4261–70.

4. Nielsen C. Animal Evolution. Interrelationships of the Living Phyla. Oxford:University Press; 2012.

5. Hyman LH. The invertebrates. Vol. V. smaller coelomate groups. New York:McGraw-Hill; 1959.

6. Helmkampf M, Bruchhaus I, Hausdorf B. Phylogenomic analyses oflophophorates (brachiopods, phoronids and bryozoans) confirm theLophotrochozoa concept. Proc R Soc B Biol Sci. 2008;275:1927–33.

7. Nesnidal M, Helmkampf M, Meyer A, Witek A, Bruchhaus I, Ebersberger I,Hankeln T, Lieb B, Struck T, Hausdorf B. New phylogenomic data supportthe monophyly of Lophophorata and an Ectoproct-Phoronid clade andindicate that Polyzoa and Kryptrochozoa are caused by systematic bias.BMC Evol Biol. 2013;13:1–13.

8. Nesnidal MP, Helmkampf M, Bruchhaus I, Ebersberger I, Hausdorf B.Lophophorata monophyletic - after all. In: Wagele JW, Bartolomaeus T,editors. Deep Metazoan Phylogeny: The Backbone of the Tree of Life: NewInsights from Analyses of Molecules, Morphology, and Theory of DataAnalysis. Berlin: Walter de Gruyter; 2014. 127–142.

9. Kocot K. On 20 years of Lophotrochozoa. Org Divers Evol. 2016;16:329–43.10. Wood TS. General features of the class Phylactolaemata. In: Robinson RA,

editor. Treatise on Invertebrate Palaeontology Part G: Bryozoa (Revised).Boulder and Lawrence: Geological Society of America and University ofKansas; 1983. p. 287–303.

11. Fuchs J, Obst M, Sundberg P. The first comprehensive molecular phylogenyof Bryozoa (Ectoprocta) based on combined analyses of nuclear andmitochondrial genes. Mol Phylogenet Evol. 2009;52:225–33.

12. Waeschenbach A, Taylor PD, Littlewood DTJ. A molecular phylogeny ofbryozoans. Mol Phylogenet Evol. 2012;62:718–35.

13. Wood TS. Phyla Ectoprocta and Entoprocta (Bryozoans). In: Thorp JH, Rogers DC,editors. Ecology and General Biology, Vol I: Thorp and Covich’s FreshwaterInvertebrates, 4th Edition. London: Academic Press; 2014. 327–345.

14. Hirose M, Dick MH, Mawatari SF. Proceedings of the 14th InternationalBryozoology Association Conference, Boone, North Carolina, July 1–8,2007, Virginia Museum of Natural History Special Publication No 15.In: Hageman SJ, Key MMJ, Winston JE, editors. Molecular phylogeneticanalysis of phylactolaemate bryozoans based on mitochondrial genesequences. Martinsville: Virginia Museum of Natural History; 2008.p. 65–74.

15. Lacourt AW. A monograph of freshwater bryozoa-Phylactolaemata. ZoolVerh Uitgeg Rijksmus Nat Hist Leiden. 1968;93:1–159.

16. Toriumi M. Taxonomical study on fresh-water Bryozoa. XI. Stephanella hinaOka. Sci Rep Tokohu Imp Univ 4e serie, Biol. 1955;21:131–6.

Schwaha et al. Zoological Letters (2016) 2:25 Page 9 of 10

17. Smith DG. Stephanella hina (Ectoprocta: Phylactolaemata) in North America, withnotes on its morphology and systematics. J N Am Benthol Soc. 1988;7:253–9.

18. Mukai H. Systematic position of Stephanella hina (Bryozoa: Phylactolaemata),with special reference to the budding site and the attachment ofsessoblasts. Zool Sci (Tokyo). 1990;7:947–54.

19. Mukai H. Comparative Morphological studies on the Statoblasts of lowerphylactolaemate bryozoans, with discussion on the Systematics of thePhylactolaemata. Sci Rep Fac Educ Gunma Univ. 1999;46:51–91.

20. Mukai H. Notes on the freshwater Bryozoa in Gunma Prerecture (centralJapan). Sci Rep Fac Educ Gunma Univ. 1982;31:17–32.

21. Oka A. Über eine neue Gattung von Süsswasserbryozoen. Annot Zool Jap.1908;6:277–85.

22. Annandale N. Preliminary note on a new genus of Phylactolaematouspolyzoa. Rec Ind Mus. 1909;3:279–80.

23. Marcus E. Sobre Bryozoa do Brasil. II. Boletim da Faculdade de filosofia,ciéncias e letras, Universidade di Sao Paolo, Zool. 1942;6:57–105.

24. Dzik J. Evolutionary relationships of the Early Paleozoic cyclostomatousBryozoa. Palaeontology. 1981;24:827–62.

25. Taylor PD, Larwood GP. Major evolutionary radiations in the Bryozoa. In: TaylorPD, Larwood GP, editors. Major evolutionary radiations. Oxford: ClarendonPress; 1990. 209–233.

26. Taylor PD. Cyclostome systematics: phylogeny, suborders and the problemof skeletal organization. In: Herrera Cubilla A, Jackson JBC, editors.Proceedings of the 11th International Bryozoology Association Conference.Balboa: Smithsonian Tropical Research Institute; 2000. p. 87–103.

27. Taylor PD. Bioimmured ctenostomes from the Jurassic and the origins ofthe cheilostome Bryozoa. Palaeontology. 1990;33:19–34.

28. Taylor PD. The ancestrula and early growth pattern in two primitivecheilostome bryozoans: Pyripora catenularia (Fleming) and Pyriporopsispartlandensis Pohowsky. J Nat Hist. 1986;20:101–10.

29. Ostrovsky AN, Taylor PD, Dick MH, Mawatari SF. Pre-CenomanianCheilostome Bryozoa: Current State of Knowledge. In: Okada H, Mawatari SF,Suzuki N, Gautam P, editors. Proceedings of International Symposium TheOrigin and Evolution of Natural Diversity, 1–5 October 2007. Sapporo:Hokkaido University; 2008. p. 69–74.

30. Mukai H, Terakado K, Reed CG. Bryozoa. In: Harrison FW, Woollacott RM, editors.Microscopic anatomy of invertebrates. Chichester: Wiley-Liss; 1997. p. 45–206.

31. Kaya M, Baublys V, Satkauskiene I, Akyuz B, Bulut E, Tubelyte V. First chitinextraction from Plumatella repens (Bryozoa) with comparison to chitins ofinsect and fungal origin. Int J Biol Macromol. 2015;79:126–32.

32. Carroll EJJ, Wei SH, Nagel GM, Ruibal R. Structure and macromolecularcomposition of the egg and embryo jelly coats of the Lepidobatrachuslaevis. Dev Growth Diff. 1991;33:37–43.

33. Gruhl A, Wegener I, Bartolomaeus T. Ultrastructure of the body cavities inPhylactolaemata (Bryozoa). J Morphol. 2009;270:306–18.

34. Tavener-Smith R, Williams A. The secretion and structure of the skeleton ofliving and fossil Bryozoa. Philos Trans R Soc Lond Ser B. 1972;264:97–159.

35. Mano R. The coelomic corpuscles and their origin in the freshwater bryozoan,Lophopodella carteri. Sci Rep Tokyo Kyoiku DaigakuSectB. 1964;11:211–35.

36. Harmer SF. On the nature of excretory processes in marine Polyzoa. Q JMicrosc Sci. 1891;33:123–67.

37. Gordon DP. The ageing process in bryozoans. In: Woollacott RM, ZimmerRL, editors. Biology of bryozoans. New York: Academic; 1977. p. 335–76.

38. Matricon I. Étude histologique d'Alcyonidium polyoum (Hassall) (BryozaireCténostome). Cah Biol Mar. 1960;1:359–95.

39. Bobin G, Prenant M. Sur les cellules cavitaires de quelques Vésicularines(Bryozaires Cténostomes). Cah Biol Mar. 1972;12:479–510.

40. Marcus E. Über Lophopus crystallinus (PALL.). Zool Jb Anat. 1934;58:501–606.41. Oda S. On the outflow of the blood in colonies of freshwater Bryozoa

[japanese]. Kagaku (tokyo). 1958;28:37.42. Gruhl A. Occurrence and Identity of “White Spots” in Phylactolaemata. In:

Ernst A, Schäfer P, Scholz J, editors. Bryozoan Studies 2010. VerlinHeidelberg: Springer; 2013. p. 91–102.

43. Wiebach F. Über den Ausstoß von Flottoblasten bei Plumatella fructicosa.Zool Anz. 1952;149:181–5.

44. Wiebach F. Über den Ausstoss von Flottoblasten bei Plumatellen. Zool Anz.1953;151:266–72.

45. Ostrovsky AN, Porter JS. Pattern of occurrence of supraneural coelomoporesand intertentacular organs in Gymnolaemata (Bryozoa) and its evolutionaryimplications. Zoomorphology. 2011;130:1–15.

46. Gruhl A, Schwaha T. Bryozoa. In: Schmidt-Rhaesa A, Harzsch S, Purschke G,editors. Structure and Evolution of Invertebrate Nervous System. Oxford:Oxford University Press; 2015. p. 325–40.

47. Gerwerzhagen A. Beiträge zur Kenntnis der Bryozoen, I. Das Nervensystemvon Cristatella mucedo. Z Wiss Zool. 1913;107:309–45.

48. Hiller S. The so-called “colonial nervous system” in Bryozoa. Nature.1939;143:1069–70.

49. Shunkina KV, Zaytseva OV, Starunov VV, Ostrovsky AN. Comparativemorphology of the nervous system in three phylactolaemate bryozoans.Front Zool. 2015;12:28.

50. Marcus E. Beobachtungen und Versuche an lebenden Süßwasserbryozoen.Zool Jb Syst. 1926;52:279–350.

51. Kraepelin K. Die deutschen Süßwasser-bryozoen. 1. Anatomisch-systematischerTeil. Abhandlungen aus dem Gebiete der Naturwissenschaften, hrsg vomNaturwissenschaftlicher Verein in Hamburg. 1887;10:168.

52. Mukai H, Oda S. Histological and histochemical studies on the epidermalsystem of higher phylactolaemate bryozoans. Annot Zool Jap. 1980;53:1–17.

53. Hirose M, Mawatari SF. Freshwater Bryozoa of Okinawa, Japan, withdescriptions of Rumarcanella gen. nov (Phylactolaemata: Plumatellidae) andtwo new species. Zootaxa. 2011;2732:1–19.

54. Wilcox AW. Locomotion in young colonies of Pectinatella magnifica. BiolBull. 1906;11:245–52.

55. Brooks CM. Notes on the statoblasts and polypides of Pectinatellamagnifica. Proc Acad Nat Sci Phila. 1929;81:427–41.

56. Mukai H, Karasawa T, Matsumoto Y. Field and laboratory studies on thegrowth of Pectinatella gelatinosa Oka, a freshwater bryozoan. Sci Rep FacEduc Gunma Univ. 1979;28:27–57.

57. Gorgoglione B, Kotob MH, El-Matbouli M. Migrating zooids allow thedispersal of Fredericella sultana (Bryozoa) to escape from unfavourableconditions and further spreading of Tetracapsuloides bryosalmonae. JInvertebr Pathol. 2016;140:97–102.

58. Koletic N, Novosel M, Rajevic N, Franjevic D. Bryozoans are returning home:recolonization of freshwater ecosystems inferred from phylogeneticrelationships. Ecol Evol. 2015;5(2):255–64.

59. Kumar G, Abd-Elfattah A, Soliman H, El-Matbouli M. Establishment ofmedium for laboratory cultivation and maintenance of Fredericella sultanafor in vivo experiments with Tetracapsuloides bryosalmonae (Myxozoa). J FishDis. 2013;36:81–8.

60. Tops S, Hartikainen H, Okamura B. The effects of infection byTetracapsuloides bryosalmonae (Myxozoa) and temperature on Fredericellasultana (Bryozoa). Int J Parasitol. 2009;39:1003–10.

61. Farmer JD. An adaptive model for the evolution of the ectoproct life cycle.In: Woollacott RM, Zimmer RL, editors. Biology of Bryozoans. New York:Academic; 1977. p. 487–518.

62. Vinn O, Ten Hove HA, Mutvei H. On the tube ultrastructure and origin ofcalcification in sabellids (Annelida, Polychaeta). Paleontology. 2008;51:295–301.

63. Vinn O, Ten Hove HA, Mutvei H, Kirsimae K. Ultrastructure and mineralcomposition of serpulid tubes (Polychaeta, Annelida). Zool J Linnean Soc.2008;154:633–50.

64. Shah DU, Vollrath F, Stires J, Deheyn DD. The biocomposite tube of achaetopterid marine worm constructed with highly-controlled orientationof nanofilaments. Mater Sci EngC Mater Biol Appl. 2015;48:408–15.

65. Herrmann K. Phoronida. In: Harrison FW, Woollacott RM, editors. Microscopicanatomy of invertebrates. Chichester: Wiley-Liss; 1997. p. 207–35.

66. Jebram D. Arguments concering the basal evolution of the Bryozoa. Z zoolSyst Evoltut-forsch. 1986;24:266–90.

• We accept pre-submission inquiries

• Our selector tool helps you to find the most relevant journal

• We provide round the clock customer support

• Convenient online submission

• Thorough peer review

• Inclusion in PubMed and all major indexing services

• Maximum visibility for your research

Submit your manuscript atwww.biomedcentral.com/submit

Submit your next manuscript to BioMed Central and we will help you at every step:

Schwaha et al. Zoological Letters (2016) 2:25 Page 10 of 10